OBJECTIVE To determine the ontogeny of Mamu-AG expression in the rhesus monkey preimplantation embryo and implantation site. RESULTS Previous studies demonstrated that the rhesus monkey placenta expresses a unique MHC class I molecule. Surprisingly, and in contrast to the human placenta (but similar to the baboon placenta), MHC class I immunostaining and mRNA expression in the definitive rhesus placenta is primarily localized to syncytiotrophoblasts in contact with maternal blood, whereas the human placenta HLA-G is localized to the extravillous cytotrophoblasts, invading maternal uterine endometrium during early gestation. We thus need to define the expression of MHC class I molecules in the earliest stages of gestation. We used PCR to evaluate the expression of Mamu-AG mRNA in preimplantation rhesus monkey embryos through the blastocyst stage. The expression of the mRNA was not detectable in these embryos, although we were able to readily amplify the mRNA for G3PDH from single embryos. The limit of sensitivity of the Mamu-AG assay is less than 10 trophoblast equivalents. However, we were able to detect Ma mu-AG mRNA and MHC class I immunostaining in trophoblasts growing out from cultured rhesus embryos, suggesting that trophoblast differentiation and outgrowth is associated with the initiation of MHC class I expression. In rhesus implantation sites at days 14, 15, 19 and 21 of gestation the trophoblastic shell (extravillous trophoblasts) were strongly positive for MHC class I immunostaining, and also demonstrated Mamu-AG expression by in situ hybridization. In these early pregnancy samples syncytiotrophoblast immunostaining was reduced in intensity compared to mature chorionic villi in the definitive placenta. These results are similar to the pattern of HLA-G expression in the human placenta during extravillous trophoblast differentiation and invasion of the maternal endometrium. FUTURE DIRECTIONS We will evaluate the expression of MHC class I molecules during embryo attachment in vivo and in vitro on defined substrates to understand the signals which give rise to developmentally regulated Mamu-AG expression. KEY WORDS trophoblast, blastocyst, endometrium, placenta, MHC. FUNDING HD34216